Mammalian cell division is controlled by a family of protein kinases known as the cyclin-dependent kinases (cdks). The goal of this project is to characterize the structural and mechanistic basis for catalysis, substrate specificity, and regulation by phosphorylation of two distinct cdk family members: cdk2/cyclinA and cdk5/p25. While cdk2/cyclinA is a classic cell division control enzyme, cdk5/p25 plays a unique role in the development of neurons - cells which classically do not divide. Activation of cdk2 typically depends on cyclin binding followed by phosphorylation. In contrast, cdk5 activation depends only on its interaction with p25, a neuronal-specific, non-cyclin-like protein. While several X-ray crystal structures of cdk2/cyclinA have been solved, there is little mechanistic information linking the abundant structural data with knowledge of this enzyme's unique substrate specificity and regulatory properties. We will describe the kinetic basis for activation of cdk2/cyclinA by phosphorylation at residue Thr160, and its inhibition by phosphorylation at Tyr15. We will describe the key structural determinants which are crucial for catalytic regulation and substrate recognition. We will compare the properties of cdk2 with those of cdk5 with an aim to understand the structural and kinetic basis for the unique regulatory properties of the cdk5/p25 complex. To achieve these goals, we will apply a variety of kinetic and biophysical techniques to isolate the individual steps of catalysis and their inherent rate constants, in both cdk2/cyclinA and cdk5/p25. We will determine which steps - substrate binding, phosphoryl group transfer, or product release - are controlled by phosphorylation and specific enzyme and substrate structural determinants. Our studies on cdk2/cyclinA and cdk5/p25 will provide the first description of how their chemical structures, catalytic mechanisms and biochemical properties are integrated, with a view to understanding the structure/function relationships of protein kinases in general.